Low affinity fcgr deficient mice

ABSTRACT

Genetically modified non-human animals and methods and compositions for making and using them are provided, wherein the genetic modification comprises a deletion of the endogenous low affinity FcγR locus, and wherein the mouse is capable of expressing a functional FcRγ-chain. Genetically modified mice are described, including mice that express low affinity human FcγR genes from the endogenous FcγR locus, and wherein the mice comprise a functional FcRγ-chain. Genetically modified mice that express up to five low affinity human FcγR genes on accessory cells of the host immune system are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/971,080, filedDec. 17, 2010, which claims the benefit of the filing date under 35 USC§119(e), and is a nonprovisional, of U.S. Provisional Patent ApplicationSer. No. 61/288,562, filed 21 Dec. 2009, which applications are herebyincorporated by reference.

FIELD OF INVENTION

The field of invention is genetically modified non-human animals thatlack endogenous murine FcγR genes, including genetically modifiedanimals that comprise a replacement of endogenous FcγR genes with humanFcγR genes, and including mice that are capable of expressing at leasttwo, three, four, or five functional human low affinity FcγR genes, andincluding genetically modified mice comprising immune cells that do notexpress endogenous low affinity FcγR genes.

BACKGROUND

Fc receptors (FcRs) are proteins found on the surface of cells of theimmune system that carry out a variety of functions of the immune systemin mammals. FcRs exist in a variety of types, on a variety of cells, andmediate a variety of immune functions such as, for example, binding toantibodies that are attached to infected cells or invading pathogens,stimulating phagocytic or cytotoxic cells to destroy microbes, orinfected cells by antibody-mediated phagocytosis or antibody-dependentcell-mediated cytotoxicity (ADCC).

ADCC is a process whereby effector cells of the immune system lyse atarget cell bound by antibodies. This process depends on prior exposureto a foreign antigen or cell, resulting in an antibody response. ADCCcan be mediated through effector cells such as, for example, naturalkiller (NK) cells, by binding of FcR expressed on the surface of theeffector cell to the Fc portion of the antibody which itself is bound tothe foreign antigen or cell. Because of the central role that FcRs playin the immune response, useful non-human animals that co-expressmultiple human FcRs are needed, including non-human animals thatco-express multiple human low affinity FcRs. There exists a need fornon-human animal models of human FcR function and human processes ofADCC for the study and elucidation of human disease therapies, inparticular anti-tumor therapies and therapies for treating autoimmunediseases, and pharmaceutical drug development, in particular in thedevelopment, design, and testing of human antibody pharmaceuticals.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depiction of a wild type low affinity FcγR locusin a mouse, showing mouse FcγRIIB, FcγRIV and FcγRIII genes and a mouseFcγR targeting vector used for a targeted deletion of these genes, whichincludes a neomycin cassette flanked by site-specific recombinationsites.

FIG. 2 shows histograms of splenocytes gated for B cells (anti-CD19), NKcells (anti-NKp46) and macrophages (anti-F4/80) including expression ofendogenous mFcγRII and mFcγRIII genes for wild type and low affinityFcγR α-chain gene-deficient mice (mFcγR KO).

FIGS. 3A-3D show in vivo depletion of B cells with a human anti-humanCD20 antibody with mouse Fc (Ab 168) or human Fc (Ab 735) in humanizedCD20 mice (hCD20) and humanized CD20 mice bred to FcγR knockout mice(hCD20/FcγR KO) in several lymphocyte compartments: bone marrow (FIG.3A), blood (FIG. 3B), lymph node (FIG. 3C) and spleen (FIG. 3D). Foreach graph, the y-axis shows the percent of gated B cells (B220⁺/IgM⁺ orB220⁺/CD19⁺) and the x-axis shows the antibody dose for each animalgroup: 10 mg/kg Control antibody (C), 2 mg/kg human anti-human CD20antibody (2 Ab) and 10 mg/kg human anti-human CD20 antibody (10 Ab).

FIG. 4 is a schematic depiction of a neomycin-targeted deletion of thelow-affinity mouse FcγR locus and a second targeting vector forinserting two human low affinity FcγR genes (hFcγRIIIA and hFcγRIIA)into the deleted mouse locus, which includes a hygromycin cassetteflanked by site-specific recombination sites. For expression of hFcγRIIAon platelets, an extended promoter region operably linked to thehFcγRIIA gene of the Human FcγRIIIA-IIA Targeting Vector is employed; toprevent expression of hFcγRIIA on platelets, the promoter region isomitted or substantially omitted.

FIG. 5A shows histograms of splenocytes gated for NK cells (anti-NKp46)and macrophages (anti-F4/80) including expression of human FcγRIIIA forwild type and human FcγRIIIA-IIA homozygote mice (Human FcγRIIIA/FcγRIIAHO).

FIG. 5B shows histograms of splenocytes gated for neutrophils(anti-Ly6G) and macrophages (anti-F4/80) including expression of humanFcγRIIA for wild type and human FcγRIIIA-IIA homozygote mice (HumanFcγRIIIA/FcγRIIA HO).

FIG. 6 is a schematic depiction of a hygromycin-targeted deletion of thelow affinity mouse FcγR locus including an insertion of two low affinityhuman FcγR genes (hFcγRIIIA and hFcγRIIA) and a third targeting vectorfor inserting three additional low affinity human FcγR genes (hFcγRIIB,hFcγRIIIB and hFcγRIIC) and a neomycin cassette flanked by site-specificrecombination sites.

FIG. 7 shows histograms of splenocytes gated for B cells (anti-CD19) andneutrophils (anti-Ly6G) including expression of human FcγRIIB and humanFcγRIIIB for wild type and human FcγRIIIA-IIIB-IIA-IIB-IIC homozygotemice (Human FcγRIIIA/FcγRIIIB/FcγRIIA/FcγRIIB/FcγRIIC HO).

SUMMARY

Genetically modified cells, non-human embryos, non-human animals andmethods and compositions for making and using them are provided. Invarious aspects, the non-human animals comprise a human FcγR receptor, adeletion of an endogenous low affinity FcγR receptor, and/or areplacement of an endogenous FcγR receptor with a human FcγR receptor atan endogenous mouse low affinity FcγR locus.

In one aspect, genetically modified cells, non-human embryos, andnon-human animals are provided that comprise a functional FcR γ-chain,wherein the cells, embryos, and animals comprise a further modificationcomprising a replacement of the low affinity endogenous non-human FcγRgene sequences (e.g., FcγRIIB, FcγRIV and FcγRIII) with one or more lowaffinity human FcγR gene sequences (e.g., selected from FcγRIIA,FcγRIIB, FcγRIIC, FcγRIIIA, FcγRIIIB, and a combination thereof).

In one embodiment, the cells, non-human embryos, and non-human animalsare murine. In one embodiment, the functional FcR γ-chain is a mouse FcRγ-chain. In one embodiment, the mouse FcR γ-chain is an FcR γ-chainendogenous to the mouse, the cell, or the embryo.

In one embodiment, the cells, embryos, and animals are mice, and themice express a functional α-chain of a human low affinity FcγR receptorand a functional endogenous mouse γ-chain.

In one aspect, a genetically modified mouse is provided, wherein themouse does not express an endogenous α-chain selected from an FcγRIIBα-chain, an FcγRIV α-chain, an FcγRIII α-chain, and a combinationthereof; wherein the mouse expresses a functional endogenous mouseγ-chain.

In a specific embodiment, the mouse does not express a functionalFcγRIIB α-chain, does not express a functional FcγRIV α-chain, and doesnot express a functional FcγRIII α-chain.

In one embodiment, the mouse genome comprises a deletion of anendogenous FcγRIIB α-chain, a deletion of an endogenous FcγRIV α-chain,and a deletion of an endogenous FcγRIII α-chain.

In one embodiment, the mouse comprises a deletion of an endogenousFcγRIIB α-chain, a deletion of an endogenous FcγRIV α-chain, and adeletion of an endogenous FcγRIII α-chain, and further comprises areduced ability to make an immune response to an antigen as comparedwith a wild type mouse's ability with respect to the same antigen. Inone embodiment, the reduced immune response includes a decreasedantibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment,the reduced immune response includes a reduced ability in a cell killingassay to achieve antibody-dependent NK cell killing. In specificembodiments, the reduction in ADCC or antibody-dependent NK cell killingis at least 50%, in one embodiment at least 75%, in one embodiment atleast 90%.

In one embodiment, the mouse comprises a deletion of an endogenousFcγRIIB α-chain, a deletion of an endogenous FcγRIV α-chain, and adeletion of an endogenous FcγRIII α-chain, and further comprises anincreased humoral antibody response upon immunization with an antigen ascompared to a wild type mouse, e.g., a mouse of the same or similarstrain that does not comprise the deletion. In one embodiment, theincreased humoral antibody response is 2-fold as compared to a wild typemouse. In one embodiment, the increased humoral antibody response is3-fold as compared to a wild type mouse. In one embodiment, theincreased humoral antibody response is 5-fold as compared to a wild typemouse. In one embodiment, the increased humoral antibody response is7-fold as compared to a wild type mouse. In one embodiment, theincreased humoral antibody response is 10-fold as compared to a wildtype mouse. In a specific embodiment, humoral antibody response ismeasured by micrograms of antibody that specifically binds an antigen(with which the mouse has been immunized) per microgram of serum proteinfrom the mouse. In one embodiment, the increased humoral antibodyresponse is with respect to an antigen to which a wild type mouseexhibits tolerance, or to an antigen which in a wild type mouse exhibitsa poor or minimal humoral immune response. In a specific embodiment, theantigen is a mouse antigen. In a specific embodiment, the antigen is ahuman antigen that exhibits an identity with a mouse protein of at leastabout 95%, 96%, 97%, 98%, or 99%.

In one aspect, a genetically modified mouse is provided, comprising areplacement of a low affinity mouse FcγR α-chain gene with a lowaffinity human FcγR α-chain gene, wherein the replacement is at theendogenous mouse FcγR α-chain gene locus. In one embodiment, the lowaffinity mouse FcγR α-chain gene is selected from an FcγRIIB, FcγRIV andan FcγRIII α-chain gene. In a specific embodiment, a geneticallymodified mouse is provided, wherein the mouse expresses an endogenousFcR γ-chain, and wherein the low affinity human FcγR α-chain gene isFcγRIIIA α-chain. In another specific embodiment, the geneticallymodified mouse expresses an endogenous FcR γ-chain and a functionalhuman FcγRIIIA α-chain on NK cells. In a specific embodiment, thefunctionality of FcγRIIIA α-chain on NK cells is reflected by humanantibody-mediated NK killing (e.g., ADCC mediated by a human antibody).

In one aspect, a genetically modified cell, non-human embryo, ornon-human animal is provided, wherein the genetic modification comprisesa replacement of at least one endogenous low affinity FcγR α-chain genewith a human FcγR α-chain gene, and the cell, embryo, or animalexpresses a functional FcR γ-chain. In one embodiment, the functionalFcRγ-chain is an endogenous FcR γ-chain. In one embodiment, the lowaffinity human FcγR α-chain gene is selected from an FcγRIIA α-chaingene, an FcγRIIIA α-chain gene, and a combination thereof. In a specificembodiment, the human FcγRIIA gene comprises a polymorphism, wherein thepolymorphism is selected from a 131His low responder polymorphism and a131Arg high responder polymorphism. In a specific embodiment, theFcγRIIA polymorphism is the 131His low responder polymorphism. In oneembodiment, the FcγRIIIA gene is a specific allelic variant, wherein theallelic variant is selected from a 158Val variant and a 158Phe variant.In a specific embodiment, the FcγRIIIA allelic variant is the 158Valvariant.

In one embodiment the low affinity human FcγR gene is selected from anFcγRIIB, FcγRIIC, an FcγRIIIB gene, and a combination thereof. In aspecific embodiment, the human FcγRIIB gene comprises an amino acidsubstitution, wherein the substitution is selected from an 232Ile or a232Thr substitution. In another specific embodiment, amino acidsubstitution is a 232Ile substitution. In a specific embodiment, theFcγRIIIB gene is a specific allelic variant, wherein the allelic variantis selected from a NA1 variant and a NA2 variant. In another specificembodiment, the FcγRIIIB allelic variant is a NA2 variant.

In one embodiment the low-affinity human FcγR α-chain gene is selectedfrom a FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, FcγRIIIB α-chain gene, and acombination thereof.

In one embodiment, the low affinity mouse FcγRIV α-chain gene and theFcγRIII α-chain gene are replaced with at least one low affinity humanFcγR α-chain gene. In one embodiment, the low affinity mouse FcγRIVα-chain gene and the FcγRIIB α-chain gene are replaced with at least onelow affinity human FcγR α-chain gene. In one embodiment, the lowaffinity mouse FcγRIIB α-chain gene and the FcγRIII α-chain gene arereplaced with at least one low affinity human FcγR α-chain gene. In aspecific embodiment, the at least one low affinity human FcγR α-chaingene is selected from an FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, FcγRIIIBα-chain gene, and a combination thereof. In another specific embodiment,the at least one low affinity human FcγR α-chain gene is selected froman FcγRIIA α-chain gene, an FcγRIIIA α-chain gene, and a combinationthereof. In another specific embodiment, the at least one low affinityhuman FcγR α-chain gene is selected from an FcγRIIB, FcγRIIC, FcγRIIIBα-chain gene, and a combination thereof. In another specific embodiment,the low affinity mouse FcγR genes are replaced with a human FcγRIIAα-chain gene and a human FcγRIIIA α-chain gene. In another specificembodiment, the low affinity human FcγRIIA and FcγRIIIA α-chain genescomprise variants, wherein the FcγRIIA α-chain gene comprises a 131Hisvariant and the FcγRIIIA α-chain gene comprises a 158Val variant. Inanother specific embodiment, the low affinity mouse FcγR α-chain genesare replaced with the following low affinity human FcγR α-chain genes:FcγRIIB, FcγRIIC and FcγRIIIB. In another specific embodiment, the lowaffinity human FcγRIIB α-chain gene and FcγRIIIB α-chain gene comprisevariants, wherein the FcγRIIB α-chain gene comprises a 232Ile variantand the FcγRIIIB α-chain gene comprises an NA2 variant.

In one embodiment, the genetic modifications comprise a replacement ofsyntenic genomic sequences of mouse and human chromosome 1. In aspecific embodiment, the genetic modifications comprise a replacement ofa genomic fragment comprising endogenous low affinity mouse FcγR geneswith a genomic fragment comprising low affinity human FcγR genes. Inanother specific embodiment, the mouse genome from chromosome1:172,889,983 to chromosome 1:172,989,911 is replaced with a humangenomic fragment comprising human chromosome 1:161,474,729 to chromosome1:161,620,458.

In one aspect, a genetically modified cell, non-human embryo, ornon-human animal is provided, wherein the genetic modification comprisesa knockout of one or more endogenous low affinity receptor α-chaingenes, and the presence of an episome comprising one or more human FcγRα-chain genes. In a specific embodiment, the cell, embryo, or animalexpresses a functional FcR γ-chain. In a specific embodiment, theepisome is a mini chromosome. In one embodiment, the functional FcRγ-chain is endogenous to the cell, embryo, or animal.

In one aspect, a genetically modified mouse is provided, comprising areplacement of a low affinity mouse FcγR α-chain gene with a lowaffinity human FcγR α-chain gene, the mouse comprises a mouse FcRγ-chaingene, and the mouse expresses a functional human low affinity FcγRreceptor. In one embodiment, the functional low affinity FcγR receptoris expressed on a cell type in which the low affinity FcγR receptor isexpressed in humans. In a specific embodiment, the functional human lowaffinity FcγR receptor is FcγRIIIA and the FcγRIIIA is expressed on NKcells.

In one embodiment, the mouse comprises a deletion of two mouse FcγRα-chain genes. In another embodiment, the mouse comprises a deletion ofthree mouse FcγR α-chain genes.

In one embodiment, the mouse comprises a replacement of three mouse FcγRα-chain genes with at least one human FcγR α-chain gene. In anotherembodiment, the mouse comprises a replacement of two mouse FcγR α-chaingenes with at least one human FcγR α-chain gene. In a specificembodiment, the mouse comprises a replacement of three mouse FcγRα-chain genes with at least two human FcγR α-chain genes. In anotherspecific embodiment, the three mouse FcγR α-chain genes are replacedwith three human FcγR α-chain genes. In another specific embodiment, themouse comprises a replacement of two mouse FcγR α-chain genes with atleast two human FcγR α-chain genes. In yet another specific embodiment,the two mouse FcγR α-chain genes are replaced with at least three humanFcγR α-chain genes.

In one embodiment, the low affinity mouse FcγR α-chain gene is selectedfrom an FcγRIIB, FcγRIV, FcγRIII α-chain gene, and a combinationthereof.

In one embodiment, the low affinity human FcγR α-chain gene is selectedfrom an FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, FcγRIIIB α-chain gene, anda combination thereof. In one embodiment, the low affinity human FcγRα-chain gene is selected from an FcγRIIA, an FcγRIIIA α-chain gene, anda combination thereof. In one embodiment, the low affinity human FcγRα-chain gene is selected from an FcγRIIB, FcγRIIC, an FcγRIIIB α-chaingene, and a combination thereof.

In one embodiment, the low affinity mouse FcγRIV α-chain gene and theFcγRIII α-chain gene are replaced with at least one human FcγR α-chaingene. In one embodiment, the low-affinity mouse FcγRIV α-chain gene andthe FcγRIIB α-chain gene are replaced with at least one human FcγRα-chain gene. In one embodiment, the low affinity mouse FcγRIIB α-chaingene and the FcγRIIIB α-chain gene are replaced with at least one humanFcγR α-chain gene. In a specific embodiment, the at least one human FcγRα-chain gene is selected from an FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA,FcγRIIIB α-chain gene, and a combination thereof. In another specificembodiment, the at least one human FcγR α-chain gene is selected from anFcγRIIA, an FcγRIIIA α-chain gene, and a combination thereof. In anotherspecific embodiment, the at least one human FcγR α-chain gene isselected from an FcγRIIB, FcγRIIC, FcγRIIIB α-chain gene, and acombination thereof. In another specific embodiment, the mouse α-chaingenes are replaced with the following human FcγR α-chain genes: FcγRIIAand FcγRIIIA. In yet another specific embodiment, the mouse α-chaingenes are replaced with the following human FcγR α-chain genes: FcγRIIB,FcγRIIC and FcγRIIIB.

In one aspect, a genetically modified mouse is provided, comprising alow affinity human FcγR α-chain and a mouse FcR γ-chain subunit, whereinthe mouse expresses the human FcγR α-chain on a cell selected from aneutrophil, an eosinophil, a basophil, a monocyte, a macrophage, aplatelet, a Langerhans cell, a dendritic cell, an NK cell, a mast cell,a B cell, a T cell, and a combination thereof. In one embodiment, themouse expresses a human FcγRIIA α-chain on a cell selected from aneturophil, a macrophage, an eosinophil, a platelet, a dendritic cell, aLangerhans cell, and a combination thereof. In one embodiment, the mouseis capable of phagocytosis, ADCC and cellular activation initiated ormediated through the expressed human FcγRIIA α-chain. In one embodimentthe mouse expresses a human FcγRIIIA α-chain on a cell selected from amacrophage, an NK cell, a monocyte, a mast cell, an eosinophil, adendritic cell, a Langerhans cell, at least one T cell type, and acombination thereof. In one embodiment, the mouse is capable of ADCCmediated through the human FcγRIIIA α-chain expressed on NK cells. In aspecific embodiment, the mouse exhibits hFcγRIIIA-mediated ADCC inresponse to an antibody comprising a human Fc.

In one embodiment, the mouse expresses both a human FcγRIIA α-chain anda human FcγRIIIA α-chain. In one embodiment, the human FcγRIIA α-chainis expressed on platelets and the human FcγRIIIA α-chain is expressed onNK cells. In one embodiment, the mouse is capable of ADCC mediated by anantibody comprising a human Fc, wherein the mediation is through eitherthe human FcγRIIA α-chain or through the human FcγRIIIA α-chainexpressed on the surface of accessory cells. In one embodiment, thehuman FcγRIIA α-chain is not expressed on platelets. In a specificembodiment wherein the human FcγRIIA α-chain is not expressed onplatelets, the mouse lacks or substantially lacks a human promotersequence that operably linked to the human FcγRIIA α-chain in a humangenome.

In one embodiment, the mouse expresses a human FcγRIIB α-chain on a cellselected from a B cell, a mast cell, a basophil, a macrophage, aneosinophil, a neutrophil, a dendritic cell, a Langerhans cell, and acombination thereof. In a specific embodiment, the mouse expresses ahuman FcγRIIB α-chain on a B cell and a mast cell. In another specificembodiment, the mouse is capable of endocytosis of immune complexesmediated through the expressed human FcγRIIB α-chain. In one embodiment,the mouse expresses a human FcγRIIC α-chain on a cell selected from aneutrophil, a macrophage, an eosinophil, a platelet, a dendritic cell, aLangerhans cell, and a combination thereof. In a specific embodiment,the mouse is capable of phagocytosis, ADCC and cellular activationinitiated through the expressed human FcγRIIC α-chain.

In one embodiment, the mouse expresses a human FcγRIIIB α-chain onneutrophils and eosinophils. In a specific embodiment, the mouse iscapable of cellular activation, phagocytosis, ADCC and degranulation,wherein the activation, phagocytosis, ADCC, and degranulation aremediated through the expressed human FcγRIIIB α-chain.

In one aspect, a mouse is provided that comprises a deletion of theendogenous FcγRIIB, FcγRIV and FcγRIII genes and insertion of humanFcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, and FcγRIIIB genes, and wherein themouse comprises a functional mouse FcR γ-chain gene.

In one embodiment, the mouse comprises a deletion of the α-chainsencoded by endogenous FcγRIIB, FcγRIV and FcγRIII genes and insertion ofthe α-chains encoded by human FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA, andFcγRIIIB genes.

In one embodiment, the insertion of the human FcγRIIA, FcγRIIB, FcγRIIC,FcγRIIIA, and FcγRIIIB α-chain genes is at a random location within themouse genome.

In one embodiment, the insertion of the human FcγRIIA, FcγRIIB, FcγRIIC,FcγRIIIA, and FcγRIIIB α-chain genes is at the endogenous mouse lowaffinity FcγR α-chain locus.

In one embodiment, the mouse expresses human FcγRIIIA on NK cells andmacrophages. In a specific embodiment, all or substantially all NK cellsfrom a splenocyte sample of the mouse express human FcγRIIIA. In aspecific embodiment, all or substantially all macrophages from asplenocyte sample of the mouse express human FcγRIIIA.

In one embodiment, the mouse expresses a human FcγR selected from humanFcγRIIA, human FcγRIIIA, and a combination thereof, on a cell typeselected from neutrophils, macrophages, and a combination thereof. In aspecific embodiment, the mouse expresses human FcγRIIA and humanFcγRIIIA on all or substantially all neutrophils and macrophages of asplenocyte sample from the mouse.

In one embodiment, the mouse expresses human FcγRIIB and human FcγRIIIBon B cells and neutrophils of B cells from a B cell-gated splenocytesample from the mouse. In a specific embodiment, the mouse expressesFcγRIIIB and FcγRIIB on all or substantially all B cells and neutrophilsfrom a B cell-gated splenocyte sample from the mouse.

In one embodiment, the mouse further comprises a humanized CD20 gene. Inone embodiment, the mouse that further comprises the humanized CD20 genefollowing treatment with an anti-CD20 binding protein that comprises anFc exhibits depletion (in vivo) of B cells. In one embodiment, thedepletion is in a compartment selected from bone marrow, blood, lymphnode, spleen, and a combination thereof. In one embodiment, the Fc is ahuman Fc. In one embodiment, the Fc is a mouse Fc. In one embodiment,the anti-CD20 binding protein is an anti-CD20 antibody.

In one aspect, a cell is provided comprising a genetic modification asdescribed herein. In one embodiment, the cell is selected from anembryonic stem (ES) cell, a pluripotent cell, an induced pluripotentcell, and a totipotent cell. In one embodiment, the cell is selectedfrom a mouse cell and a rat cell. In a specific embodiment, the cell isan ES cell. In a more specific embodiment, the cell is a mouse ES cell.

In one aspect, a non-human embryo is provided, comprising a geneticmodification as described herein. In one embodiment, the non-humanembryo is selected from a mouse embryo and a rat embryo.

In one aspect, a method is provided for determining efficacy of atherapeutic. In one embodiment, the therapeutic is an antibody (e.g.,mono-, bi-, tri-, multispecific) comprising a human Fc. In oneembodiment, the therapeutic is a human antibody. In one embodiment, theefficacy is efficacy of therapeutic-mediated cell killing (e.g., ADCC).In a specific embodiment, the human therapeutic is a fusion proteincomprising an Fc of a human immunoglobulin heavy chain. In oneembodiment, the therapeutic is administered to a mouse as describedherein and a level of therapeutic-dependent ADCC is measured. In oneembodiment, the mouse is used to assess the ADCC activity of atherapeutic by administering the therapeutic to the mouse and thendetecting (e.g., in vitro from a sample (e.g., blood) taken from theanimal) binding of the therapeutic to a human low affinity FcγR on anFcγR-expressing cell. In a specific embodiment, accessory cells of themouse are isolated from the mouse and tested for the ability, in thepresence and absence of the therapeutic, to mediatetherapeutic-dependent ADCC.

In one aspect, a method is provided for determining whether a lowaffinity FcγR is associated with a human disease or disorder, comprisinga step of determining a trait associated with the human disease ordisorder in a mouse according to the invention. In one embodiment, thetrait is a phenotype associated with the absence or loss of a functionof one or more low affinity FcγRs. In a specific embodiment, the diseaseor disorder is an autoimmune disease or disorder. In a specificembodiment, the autoimmune disease or disorder is selected fromRheumatoid Arthritis (RA), Systemic Lupus Erythematosus (SLE), type Idiabetes, Guillain-Barré syndrome, sclerosis, multiple sclerosis,Goodpasture's syndrome, Wegener's Granulomatosis and experimentalautoimmune encephalomyelitis (EAE). In a specific embodiment, the mousecomprises a polymorphism in a low affinity FcγR, and the trait isselected from an enhanced ability to mediate ADCC in comparison to themajority of the human population that does not bear the polymorphism,and a reduced ability to mediate ADCC in comparison to the majority ofthe human population that does not bear the polymorphism.

In one aspect, a method for making an anti-human FcR α-chain antibody ina mouse is provided, comprising exposing a mouse according to theinvention to a human FcR as described herein. In one embodiment, anantibody that recognizes the human FcR is isolated from the mouse. Inanother embodiment, a nucleic acid sequence that encodes all or part ofa variable region of an antibody that recognizes the human FcR isidentified and cloned.

In one aspect, a method for determining ability of anti-human FcRantibodies to target molecules to FcR-expressing cells for phagocytosisof the target molecule is provided, comprising exposing a mouse asdescribed herein to an agent comprising an anti-human FcR antibody, andmeasuring phagocytosis of the target molecule.

In one aspect, a method is provided for making an antibody, in a mouse,to an antigen that is poorly immunogenic in a mouse that is wild typewith respect to one or more FcγRs, comprising exposing a mouse asdescribed herein that lacks a mouse low affinity FcR but expresses anFcγR γ-chain to the antigen that is poorly immunogenic in the mouse thatis wild type with respect to one or more FcγRs, and identifying anantibody that recognizes the poorly antigenic antigen. In oneembodiment, the method comprises isolating the antibody from the mouse.In another embodiment, a nucleic acid sequence that encodes all or partof a variable region of the antibody is identified and cloned.

In one aspect, a method for making a mouse capable of making antibodiescomprising human variable regions is provided, comprising a step ofbreeding a first mouse as described herein with a second mouse thatcomprises (a) one or more human immunoglobulin variable region genesegments and one or more human constant region genes; or, (b) one ormore human immunoglobulin variable region gene segments operably linkedto a mouse constant region gene, wherein the human gene segments replacevariable region gene segments at the mouse variable region gene segmentlocus.

In one embodiment, the second mouse (a) comprises a transgene thatcomprises one or more human immunoglobulin light chain variable regiongene segments and a human light chain constant gene, and a transgenethat comprises one or more human immunoglobulin heavy chain variableregion gene segments and one or more human heavy chain constant genes.In one embodiment, the transgene that comprises one or more humanimmunoglobulin heavy chain variable region gene segments comprises twoor more heavy chain constant genes and is capable of class switching. Ina specific embodiment, the mouse comprises an inactivated endogenouslight chain locus and/or an inactivated endogenous heavy chain locus. Ina specific embodiment, the mouse comprises a deletion of an endogenouslight chain locus and/or a deletion of an endogenous heavy chain locus.

In one embodiment, the second mouse (b) comprises human heavy and humanlight variable region gene segments, at the heavy an light mouse loci,respectively.

In one aspect, a method is provided for selecting an anti-tumorantibody, comprising a step of determining the ability of an antibody tomediate ADCC, wherein the ability of the antibody to mediate ADCC istested by determining ADCC mediated by a cell of a mouse as describedherein, and the antibody is selected if it mediates ADCC employing acell of a genetically modified mouse as described herein. In a specificembodiment, binding of the antibody to the cell of the geneticallymodified mouse is determined, and the anti-tumor antibody is selectedfor its ability to bind a human FcγR on the cell. In a specificembodiment, the human FcγR is a low affinity FcγR.

In one embodiment, the anti-tumor antibody is identified by its enhancedability to mediate ADCC through a cell of the mouse as compared toability of the anti-tumor antibody to mediate ADCC through a cell of awild type mouse. In a specific embodiment, the anti-tumor antibody isidentified by its ability to mediate ADCC through NK cells. In aspecific embodiment, the NK cells express human FcγRIIIA.

In one embodiment, a method is provided for selecting an anti-tumoragent, comprising a step of administering an agent comprising a human Fcor a modified human Fc to a first non-human animal wherein the firstnon-human animal is genetically modified in accordance with theinvention and comprises a human tumor; a step of administering the agentto a second non-human animal comprising the tumor; and determining theability of the first non-human animal and the second non-human animal toretard growth of the human tumor following administration of the agent,wherein the agent is selected as an anti-tumor agent if it exhibits anenhanced ability to retard growth of the human tumor in the firstnon-human animal but not in the second non-human animal.

In one embodiment, the first non-human animal is modified to comprise adeletion of an endogenous FcR α-subunit, and is modified to comprise ahuman FcR α-subunit selected from the group consisting of an FcγRIIAα-subunit, an FcγRIIB α-subunit, an FcγRIIC α-subunit, an FcγRIIIAα-subunit, an FcγRIIIB α-subunit, and a combination thereof. In oneembodiment, the second animal is a wild type animal. In one embodiment,the first non-human animal expresses an endogenous FcR γ-chain.

In one embodiment, the first non-human animal expresses a functionalendogenous FcγRI.

In one aspect, a method is provided for making a mouse that lacks a lowaffinity mouse FcγR, expresses a functional FcR γ-chain, and comprisesgenes encoding α-chains of the human FcγRIIA, FcγRIIB, FcγRIIC,FcγRIIIA, and FcγRIIIB, comprising a step of replacing the low affinitymouse FcγR α-chains with human FcγR α-chains, at the mouse FcγR α-chainlocus.

In one embodiment, a first step comprises deleting the α-chains of theendogenous FcγRIIB, FcγRIV and FcγRIII genes and inserting the α-chainsof the human FcγRIIA and FcγRIIIA genes; a second step comprisesinserting the α-chains of the human FcγRIIB, FcγRIIC and FcγRIIIB genesinto the mouse genome that results from the first step; wherein themouse comprises a functional mouse FcR γ-chain gene. In a specificembodiment, the α-chains of the human FcγRIIB, FcγRIIC and FcγRIIIBgenes of the second step are inserted 5′ relative to the α-chains of thehuman FcγRIIA and FcγRIIIA genes of the first step.

In one aspect, a method for determining cell killing by a humantherapeutic in a non-primate is provided, comprising a step of exposinga cell, non-human embryo, or non-human animal to a human therapeuticthat comprises a human Fc, wherein the cell, embryo, or animal comprisesa functional FcR γ-chain and comprises a replacement of one or moreendogenous low affinity FcγR α-chain genes with one or more human FcγRα-chains, and determining the ability of the human therapeutic tomediate cell killing through a low affinity human FcγR of the cell,embryo, or animal.

In one embodiment, the non-primate is a mouse. In a specific embodiment,endogenous mouse FcγR α-chain genes FcγRIIB, FcγRIV and FcγRIII arereplaced with human FcγR α-chain genes FcγRIIA, FcγRIIB, FcγRIIC,FcγRIIIA, and FcγRIIIB.

In one embodiment, the cell is selected from a B cell, a mast cell, abasophil, a macrophage, an eosinophil, a neutrophil, a dendritic cell, aLangerhans cell, and a combination thereof. In a specific embodiment,the cell is an NK cell and NK cell-mediated ADCC by a human or ahumanized antibody is determined. In a specific embodiment, the lowaffinity human FcγR is a human FcγRIIIA.

In one aspect, a method for determining therapeutic-dependent thrombosisis provided, comprising exposing a first non-human animal that expressesa human FcγRIIA on a platelet to a therapeutic; exposing a secondnon-human animal that does not express the human FcγRIIA on a plateletto said therapeutic; measuring in the first non-human animal and in thesecond non-human animal an amount of therapeutic-dependent thrombosis;and, determining a difference in therapeutic-dependent thrombosis.

In one embodiment, the non-human animal is selected from a mouse and arat.

In one embodiment, the determined difference in therapeutic-dependentthrombosis is employed to identify a risk associated with administeringthe therapeutic to a human. In one embodiment, the determined differenceresults in a change of administration of the therapeutic to a humanpatient in need thereof.

DETAILED DESCRIPTION

The invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. Theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

The phrase “targeting construct” includes a polynucleotide molecule thatcomprises a targeting region. A targeting region comprises a sequencethat is substantially homologous to a sequence in a target cell, tissueor animal and provides for integration of the targeting construct into aposition within the genome of the cell, tissue or animal. In a specificembodiment, the targeting construct further comprises a nucleic acidsequence or gene of particular interest, a selectable marker, controland or regulatory sequences, and other nucleic acid sequences that allowfor recombination mediated through the exogenous addition of proteinsthat aid in or facilitate recombination involving such sequences. Inanother specific embodiment, the targeting construct further comprises agene of interest, wherein the gene of interest is a heterologous genethat encodes a protein that has a similar function as a protein encodedby the endogenous sequence.

The term “replacement” includes wherein a DNA sequence is placed into agenome of a cell in such a way as to replace a sequence within a genome,at the locus of the genomic sequence, with a heterologous sequence(e.g., a human sequence in a mouse), unless otherwise indicated. The DNAsequence so placed may include one or more regulatory sequences that arepart of source DNA used to obtain the sequence so placed (e.g.,promoters, enhancers, 5′- or 3′-untranslated regions, etc.). Forexample, in various embodiments, the replacement is a substitution of anendogenous sequence for a heterologous sequence that results in theproduction of a gene product from the DNA sequence so placed (comprisingthe heterologous sequence), but not expression of the endogenoussequence; the replacement is of an endogenous genomic sequence with aDNA sequence that encodes a protein that has a similar function as aprotein encoded by the endogenous genomic sequence (e.g., the endogenousgenomic sequence encodes a low affinity mouse FcγR receptor, and the DNAfragment encodes one or more human low affinity FcγR receptors, such as,e.g., a human FcγRIIC and/or an FcγRIIIB).

The term “FcγR” includes a receptor for an Fc, e.g., an Fc portion of anIgG immunoglobulin. The FcγR genes include an α-chain that is expressedon the surface of the cell and serves as a ligand-binding domain, andassociates with either a homodimer of the FcR γ-chain or a heterodimerof the FcR γ-chain and the 6-chain. There are several different FcγRgenes and they can be categorized into low affinity and high affinitytypes according to preferential binding to IgG in immune complexes. Lowaffinity FcγR genes in humans include FcγRIIA, FcγRIIB, FcγRIIC,FcγRIIIA and FcγRIIIB and within most of these genes naturally occurringgenetic differences, or polymorphisms, have been described in humansubjects with autoimmune diseases. Persons of skill upon reading thisdisclosure will recognize that one or more endogenous low affinity FcγRgenes in a genome (or all) can be replaced by one or more heterologouslow affinity FcγR genes (e.g., variants or polymorphisms such as allelicforms, genes from another species, chimeric forms, etc.).

The phrase “allelic variants” includes variations of a normal sequenceof a gene resulting in a series of different forms of the same gene. Thedifferent forms may comprise differences of up to, e.g., 20 amino acidsin the sequence of a protein from a gene. For example, alleles can beunderstood to be alternative DNA sequences at the same physical genelocus, which may or may not result in different traits (e.g., heritablephenotypic characteristics) such as susceptibility to certain diseasesor conditions that do not result in other alleles for the same gene orresult in varying degrees in the other alleles.

An “accessory cell” includes an immune cell that is involved in theeffector functions of the immune response. Exemplary immune cellsinclude a cell of lymphoid or myeloid origin, e.g., lymphocytes, naturalkiller (NK) cells, monocytes, macrophages, neutrophils, eosinophils,basophils, platelets, Langerhans cells, dendritic cells, mast cells etc.Accessory cells carry out specific functions of the immune systemthrough receptors, e.g., FcRs, expressed on their surfaces. In aspecific embodiment, an accessory cell is capable of triggering ADCCmediated through an FcR, e.g., a low affinity FcγR, expressed on thecell surface. For example, macrophages expressing FcRs are involved inphagocytosis and destruction of antibody-coated bacteria. Accessorycells might also be capable of releasing an agent that mediates otherimmune processes. For example, mast cells can be activated by antibodybound to FcRs to release granules, e.g., inflammatory molecules (e.g.,cytokines) at a site of infection. In various other embodiments, theexpression of FcRs on accessory cells can be regulated by other factors(e.g., cytokines). For example, FcγRI and FcγRIII expression can beinducted by stimulation with interferon-γ (IFN-γ).

Mouse and Human FcRs

The receptors for the Fc (i.e., constant) regions of immunoglobulins(FcRs) play an important role in the regulation of the immune response.FcRs are present on accessory cells of the host's immune system toeffectively dispose of foreign antigens bound by an antibody. FcRs alsoplay important roles in balancing both activating and inhibitoryresponses of the accessory cells of the immune system. FcRs are involvedin phagocytosis by macrophages, degranulation of mast cells, uptake ofantibody-antigen complexes and modulation of the immune response, aswell as other immune system processes.

In mice and humans, distinct FcRs are differentially expressed on thesurface of different accessory cells that are each specific for theimmunoglobulin isotypes present in the expressed antibody repertoire.For example, immunoglobulin G (IgG) antibodies mediate effectorfunctions through IgG receptors (FcγRs). FcγRs have been classified intothree groups: high affinity activating FcγRI (CD64), low affinityinhibitory FcγRII (CD32) and low affinity activating FcγRIII (CD16).Although each group is present in both mice and humans, the number ofisoforms and subsets of immune cells on which they are present aredifferent. For example, FcγRIIA and FcγRIIIB are expressed on accessorycells in humans but are reportedly absent from mice. Further, affinitiesof the different IgG isotypes (e.g., IgG1) for each FcγR is different inmice and humans.

Activation or inhibition of cell signaling through FcγRs and theeffector functions associated with antibody binding to FcγRs arebelieved to be mediated by specific sequence motifs of intracellulardomains of FcγRs, or of the subunits of co-receptors. Activatingreceptors are most commonly associated with the common γ-chain (FcRγ-chain) which contains an immunoreceptor tyrosine-based activationmotif (ITAM). ITAMs contain a specific sequence of about 9-12 aminoacids that include tyrosine residues that are phosphorylated in responseto antibody binding to an FcR. Phosphorylation leads to a signaltransduction cascade. Mice that lack a gene encoding an FcR γ-chain (FcRγ-chain KO) have been reported (e.g., see Takai et al. (1994) FcR γChain Depletion Results in Pleiotrophic Effector Cell Defects, Cell76:519-529; van Vugt et al. (1996) FcR γ-Chain Is Essential for BothSurface Expression and Function of Human FcγRI (CD64) In Vivo, Blood87(9):3593-3599; and Park et al. (1998) Resistance of FcReceptor-deficient Mice to Fatal Glomerulonephritis, J. Clin. Invest.102(6):1229-1238). The FcR γ-chain is reportedly essential for propersurface expression and function (e.g., signal transduction,phagocytosis, etc.) of most of the FcRs; FcR γ-chain KO mice lack FcγRIaccording to some reports. However, other reports reveal that FcRγ-chain KO mice indeed express FcγRI on the surface of certain accessorycells, and the FcγRI expressed reportedly appears functional in that itbinds IgG in mice in the absence of expressed FcR γ-chain (Barnes et al.(2002) FcγRI-Deficient Mice Show Multiple Alterations to Inflammatoryand Immune Responses, Immunity 16:379-389).

In contrast, FcγRIIB is an inhibitory receptor that contains animmunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmicdomain. Like ITAMs, ITIMs are sequence motifs that includephosphorylatable tyrosine residues. However, downstream events followingphosphorylation of an ITM lead to inhibition, not activation, of immunecell functions. Mice deficient in FcγRIIB reportedly exhibit anincreased antibody response in comparison to wild type mice (Takai etal. (1996) Augmented humoral and anaphylactic responses inFcγRII-deficient mice, Nature 379:346-349), an observation that supportsthe role of FcγRIIB as a downregulator of the B cell antibody response.

In humans, FcγRIIA, FcγRIIB, FcγRIIC, FcγRIIIA and FcγRIIIB areconsidered the classical low affinity FcγR genes and are locatedtogether on the same chromosome (Su et al. (2002) Genomic organizationof classical human low-affinity Fcγ receptor genes, Genes and Immunity 3(Supple 1):S51-S56). These genes exhibit several polymorphismsassociated with distinct phenotypes, e.g., an alteration of ligandbinding and function of the receptor. Some polymorphisms are associatedwith autoimmune diseases, e.g., systemic lupus erythematosus (SLE),rheumatoid arthritis (RA), and multiple sclerosis (MS). Transgenic micefor different human FcγRs (hFcγRs) have been developed and used asdisease models, generating high affinity antibodies, testing therapeuticantibodies for ability to elicit specific cellular responses, screeningcompounds that ameliorate aberrant immune responses, etc. (e.g., seeHeijnen et al. (1996) A Human FcγRI/CD64 Transgenic Model for In VivoAnalysis of (Bispecific) Antibody Therapeutics, J. Hematother.4:351-356; Heijnen and van de Winkel (1996) Antigen Targeting toMyeloid-specific Human FcγRI/CD64 Triggers Enhanced Antibody Responsesin Transgenic, J. Clin. Invest. 97(2):331-338; U.S. Pat. Nos. 6,111,166,6,676,927, 7,351,875, 7,402,728, and 7,416,726).

Despite the significant roles of the FcRs in providing the bridgebetween antibodies and accessory cells of the immune system, no modelsystem currently exists in which all the low affinity hFcγRs areexpressed. A mouse in which all the low-affinity hFcγRs areco-expressed—including mice that lack endogenous mouse FcγRs—in variousembodiments could be used to accurately reflect effects of a humanantibody therapeutic, including ADCC-mediated effects. Such a mousewould serve as a vital tool in the engineering, analysis and evaluationof therapeutic antibodies for treatment of human diseases such as, e.g.,RA, type I diabetes, SLE, and autoimmunity, by providing an animal modelcapable of achieving a more accurate assessment of immunologicalprocesses in humans, particularly in the context of testing humanantibody therapeutics. The mouse will also be a valuable source of cellsbearing the low affinity receptors, which cells can be used in in vitroassays for assessing therapeutic-dependent cell killing for therapeuticsthat bind the low affinity receptors, and thus for identifying usefulhuman therapeutics.

Endogenous Low Affinity FcγR Gene Deficient Mice

Genetically modified non-human animals are provided that do not expressendogenous low affinity mouse FcγR genes, but that express an endogenousmouse FcR γ-chain. In various embodiments, the FcR γ-chain is expressedin a distribution (i.e., in cell types) and at a level in the mouse thatis the same or substantially the same as in a wild type mouse.Endogenous low affinity FcγR genes can be expressed either on thesurface of immune cells or in a soluble manner in the periphery of theanimals. Genetic modifications for making a non-human animal that doesnot express endogenous low affinity mouse FcγR genes are convenientlydescribed by using the mouse as an illustration. A genetically modifiedmouse according to the invention can be made in a variety of ways,particular embodiments of which are discussed herein.

A schematic illustration (not to scale) of low affinity mouse FcγR genelocus is provided in FIG. 1 (top) to show FcγR gene arrangement at theendogenous locus. As illustrated, low affinity mouse FcγR genes FcγRIIB,FcγRIV and FcγRIII are present together in close proximity on onechromosome. Each of these genes comprise the α-chain or ligand bindingdomain responsible for the binding the Fc portion of an antibodymolecule.

A genetically modified mouse lacking a nucleotide sequence encoding anα-chain of the endogenous low affinity FcγR genes can be made by anymethod known in the art. For example, a targeting vector can be madethat deletes the low affinity mouse FcγR α-chain genes with selectablemarker gene. FIG. 1 illustrates a mouse genome (bottom) targeted by atargeting construct having a 5′ homology arm containing sequenceupstream of the endogenous low affinity FcγR α-chain locus, followed bya drug selection cassette (e.g. a neomycin resistance gene flanked byloxP sequences), and a 3′ homology arm containing sequence downstream ofthe endogenous low affinity FcγR α-chain locus. Upon homologousrecombination at the locus, the endogenous low affinity FcγR α-chainlocus is replaced by a drug selection cassette (bottom of FIG. 1). Theendogenous low affinity FcγR α-chain gene locus is thereby deletedresulting in a cell or non-human animal that does not express endogenouslow-affinity mouse FcγR α-chain genes. The drug selection cassette mayoptionally be removed by the subsequent addition of a recombinase (e.g.,by Cre treatment).

Genetically modifying a mouse to render an endogenous low-affinity mouseFcγR α-chain gene or genes nonfunctional, in various embodiments,results in a mouse that exhibits defects in immune responses, making themouse useful for evaluating cooperative, as well as individual, roles ofthe endogenous low-affinity mouse FcγR genes in normal and disorderedimmune function, IgG-mediated processes, and autoimmune disease. Invarious embodiments, modifying the α-chains of the endogenouslow-affinity mouse FcγR genes, but not the FcR γ-chain, avoids apotential reduction of other endogenous FcR genes (e.g., high affinityFcγRI) that require the FcR γ-chain for surface expression and function,thus maintaining various other immunological functions and processesmediated through γ-chain-dependent processes.

According to some reports, FcR γ-chain deficient mice lack surfaceexpression of FcγRIII and FcγRI. However, FcγRI has reportedly beendetected on the cell surface in FcR γ-chain deficient mice and isreportedly at least partially functional. In contrast, mice according tothe present invention contain unmodified endogenous FcR γ-chain, whichpreserves natural cell surface expression patterns and cellularfunctions of other FcR genes that require FcR γ-chain.

In various embodiments, mice of the present invention present anadvantage over other FcγR gene-deficient mice in that the geneticmodifications that they bear result in the maintenance of other genesnecessary for other immunological functions not entirely devoted to lowaffinity FcγR genes. For example, with a functional FcR γ-chain, otherγ-chain-dependent proteins (e.g., FcγRI) will be able to associate withthe FcR γ-chain and participate in effector cell functions in the immuneresponse. In various genetically modified mice in accordance with theinvention, it is believed that maintaining such functions (due to thepresence of a functional FcRγ-chain) while deleting endogenous lowaffinity FcγR genes (one or more α-subunits) enables a more preciseelucidation of the roles of FcRs in autoimmunity.

Low Affinity FcγR Humanized Mice

Genetically modified non-human animals are provided that expresslow-affinity human FcγR genes. Low affinity human FcγR genes can beexpressed either on the surface of accessory cells of the animal'simmune system or in a soluble manner in the periphery of the animals.

The genetic modification, in various embodiments, comprises a deletionof a functional α-chain of one or more low-affinity mouse FcγR genes,and in some embodiments a further modification comprising a replacementwith two or more, with three or more, with four or more, or with fivelow-affinity human FcγR α-subunit genes, wherein the non-human animalexpresses a functional mouse FcR γ-chain gene. Genetically modifiednon-human embryos, cells, and targeting constructs for making thenon-human animals, non-human embryos, and cells are also provided.

Compositions and methods for making a mouse that expresses a human FcγRgene, including specific polymorphic forms or allelic variants (e.g.,single amino acid differences), are provided, including compositions andmethod for making a mouse that expresses such genes from a humanpromoter and a human regulatory sequence. The methods includeselectively rendering an endogenous low affinity mouse FcγR genenonfunctional (e.g., by a deletion of its α-chain), and employing anα-chain of a low affinity human FcγR gene at the endogenous low affinitymouse FcγR gene locus to express a low affinity human FcγR α-subunitgene in a mouse. The deletion of the low affinity mouse FcγR gene ismade by deletion of one or more α-chain genes, but not an FcRγ-chaingene. The approach selectively renders one or more endogenous lowaffinity FcγR α-chain genes nonfunctional while retaining a functionalendogenous FcRγ-chain.

The endogenous FcγR α-chain replacement approach employs a relativelyminimal disruption in natural FcγR-mediated signal transduction in theanimal, in various embodiments, because the genomic sequence of the FcγRα-chains are replaced in a single fragment and therefore retain normalfunctionality by including necessary regulatory sequences. Thus, in suchembodiments, the FcγR α-chain modification does not affect otherendogenous FcRs dependent upon functional FcRγ-chain molecules. Further,in various embodiments, the modification does not affect the assembly ofa functional receptor complex involving an FcγR α-chain and theendogenous FcR γ-chain, which is believed to be required for properexpression of some FcγR α-chains on the cell surface and for downstreamsignaling resulting from an activated receptor. Because the FcR γ-chainis not deleted, in various embodiments animals containing a replacementof endogenous FcγR α-chain genes with human FcγR α-chain genes should beable to process normal effector functions from antibodies throughbinding of the Fc portion of IgG immunoglobulins to the human FcγRα-chains present on the surface of accessory cells.

A schematic illustration (not to scale) of a deleted endogenous lowaffinity mouse FcγR gene is provided in FIG. 4 (top). As illustrated,low affinity human FcγR genes FcγRIIA and FcγRIIIA are inserted into thedeleted endogenous low affinity mouse FcγR gene locus by a targetingconstruct (Human FcγRIIIA-IIA Targeting Vector) with a genomic fragmentcontaining the human low affinity human FcγRIIA and FcγRIIIA genes. Eachof these genes comprise the α-chain or ligand-binding domain of thehuman FcγR genes responsible for the binding the Fc portion of anantibody molecule.

A genetically modified mouse that expresses low affinity human FcγRgenes at the endogenous low affinity mouse FcγR locus can be made by anymethod known in the art. For example, a targeting vector can be madethat introduces low affinity human FcγR genes (e.g., FcγRIIA andFcγRIIIA) with a selectable marker gene. FIG. 4 illustrates a mousegenome comprising a deletion of the endogenous low affinity FcγR locus(top). As illustrated, the targeting construct contains a 5′ homologyarm containing sequence upstream of the endogenous low affinity mouseFcγR locus, followed by a drug selection cassette (e.g., a hygromycinresistance gene flanked on both sides by loxP sequences), a genomicfragment containing a human FcγRIIA gene, human HSP76 gene and humanFcγRIIIA gene, and a 3′ homology arm containing sequence downstream ofthe endogenous low affinity mouse FcγR locus. Upon homologousrecombination at the deleted locus, the drug selection cassette isreplaced by the sequence contained in the targeting vector (bottom ofFIG. 4). The endogenous low affinity FcγR gene locus is thus replacedwith low affinity human FcγR genes resulting in a cell or animal thatexpresses low-affinity human FcγR genes. The drug selection cassette mayoptionally be removed by the subsequent addition of a recombinase (e.g.,by Cre treatment).

For expression of hFcγRIIA on platelets, the targeting construct HumanhFcγRIIA-IIA Targeting Vector comprises an extended sequence thatincludes, e.g., all or substantially all of the human promoter regionoperably linked to the hFcγRIIA gene in a human genome. For preventingexpression of hFcγRIIA on platelets, the targeting construct lacks allor substantially all of the human promoter region operably linked to thehFcγRIIA gene in a human.

Further modifications to the chimeric locus (bottom of FIG. 4) can beachieved using similar techniques as described for replacement with twohuman FcγR genes. The modification to replace the endogenous lowaffinity FcγR gene locus with two human FcγR genes can further provide astarting point for incorporation of other low affinity human FcγR genes.For example, a schematic illustration (not to scale) of an endogenouslow affinity FcγR locus replaced with two human low affinity FcγR genesis provided in FIG. 6 (top). As illustrated, low affinity human FcγRgenes FcγRIIB, FcγRIIC and FcγRIIIB are inserted into the modifiedendogenous low affinity mouse FcγR gene locus by another targetingconstruct (Human FcγRIIB-IIIB-IIC Targeting Vector) with a genomicfragment containing the low affinity human FcγRIIB, FcγRIIC and FcγRIIIBgenes. Each of these genes comprise the α-chain or ligand-binding domainof the human FcγR genes responsible for the binding the Fc portion of anantibody molecule.

A genetically modified mouse that expresses five low affinity human FcγRgenes at the endogenous low affinity mouse FcγR locus can be made by anymethod known in the art. For example, a targeting vector can be madethat introduces low affinity human FcγR genes (e.g., FcγRIIB, FcγRIICand FcγRIIIB) with a selectable marker gene. FIG. 6 illustrates a mousegenome comprising a replacement of the endogenous low affinity FcγRlocus with two low affinity human FcγR genes (top). As illustrated, thetargeting construct contains a 5′ homology arm containing sequenceupstream of the endogenous low affinity mouse FcγR locus, followed by adrug selection cassette (e.g., a neomycin resistance gene flanked onboth sides by loxP sequences), a genomic fragment containing a humanFcγRIIB gene, a human FcγRIIIB, a human HSP77 gene, a human FcγRIICgene, followed by a 3′ homology arm containing sequence upstream of thelow affinity human FcγRIIIA gene present at the endogenous locus. Uponhomologous recombination at the modified locus, a human FcγRIIB,FcγRIIIB and FcγRIIC gene are inserted 5′ to the human FcγRIIIA andFcγRIIA genes previously present at the endogenous low affinity FcγRgene locus by the sequence contained in the targeting vector (bottom ofFIG. 6). The modified endogenous low affinity FcγR gene locus is thusfurther modified to incorporate three additional low affinity human FcγRgenes resulting in a cell or animal that expresses five low-affinityhuman FcγR genes. The drug selection cassette may optionally be removedby the subsequent addition of a recombinase (e.g., by Cre treatment).FIG. 6 (bottom) shows the structure of the resulting locus, which willexpress five low affinity human FcγR genes that can be detected on thesurface of accessory cells of the animal's immune system andindependently associate, as appropriate, with an endogenous FcRγ-chain.

Experimental Models of FcγR Deficient Mice and FcγR Humanized Mice

Genetically modified non-human animals that do not express endogenouslow affinity mouse FcγR genes are useful, e.g., to elucidate the variousfunctions of the individual low affinity FcγR genes in the immuneresponse, to measure the efficacy of a human therapeutic antibody viacell-mediated immunity (e.g., ADCC), to determine an FcγR's role inimmune diseases or disorder, to serve as models of immune diseases ordisorders, to generate antibodies against one or more FcγR proteins, andto serve as breeding mates to generate other genetically modified miceof interest.

In one embodiment, a mouse according to the invention can be used todetermine a cytotoxic effect lost (in comparison to a wild type mouse)by a mouse that does not express low affinity FcγR genes byadministering an agent to such a mouse, where the agent is known totrigger an FcγR-dependent cytotoxic effect in wild type mice. In oneembodiment, a mouse of the present invention is implanted with tumorcells and, after a subsequent period of time, injected with an antibodyspecific for an antigen expressed on the surface of the tumor cells. Theisotype of the antibody is known prior to injection and the animals areanalyzed for impairment of FcγR-dependent ADCC by comparison to ADCCobserved in wild type animals.

In another aspect, mice deficient in endogenous low affinity receptorscould be combined (e.g., by breeding) with other immune deficient miceto develop in vivo models of autoimmune disease. For example, SevereCombined Immunodeficiency (SCID) mice are routinely used in the art asmodel organisms for studying the immune system. SCID mice have animpaired ability to make T or B lymphocytes, or activate some componentsof the complement system, and cannot efficiently fight infections,reject tumors, and reject transplants. Low affinity FcγR α-subunitgene-deficient mice of the present invention may be bred to SCID mice toascertain cell depletion in a host animal in response to administrationof an antibody therapeutic (e.g., an anti-tumor antibody), which woulddetermine the roles of ADCC and complement-dependent cytotoxicity (CDC)in tumor cell depletion in vivo.

In another aspect, genetically modified non-human animals comprising areplacement of the endogenous low affinity FcγR genes with low-affinityhuman FcγR genes are provided. Such animals are useful for studying thepharmacokinetics of fully human antibodies and hFcγR-mediated ADCC. Inaddition, human FcγR genes have been shown to exhibit polymorphisms orallelic variants associated with disease (e.g., SLE, RA, Wegener'sgranulomatosis, Guillain-Barré syndrome and Multiple Sclerosis). Thus,genetically modified non-human animals that comprise a replacement ofthe endogenous low affinity FcγR genes with specific allelic orpolymorphic forms of human FcγR genes can be used to study humanautoimmune diseases, and traits associated with the polymorphisms, inthe animal. In a specific embodiment, the allelic forms of human FcγRgenes are associated with enhanced efficacy for human IgG.

In another specific embodiment, the affect of a human low affinity FcγRpolymorphism on the efficacy of a human antibody therapeutic isdetermined. In a specific embodiment, an anti-tumor antibody isadministered to a first humanized mouse comprising a first polymorphismof a human FcγR and also to a second humanized mouse comprising a secondpolymorphism of a human FcγR, wherein the first and the second mice eachcomprise a human tumor cell; and the anti-tumor activity of theanti-tumor antibody is assessed in the first mouse and in the secondmouse. In a specific embodiment, a treatment option is selected by aphysician with respect to treating a human having the first or thesecond polymorphism and having a tumor corresponding to the human tumorcell, based on the assessment of efficacy of the anti-tumor antibody inthe first mouse and in the second mouse.

Suitable polymorphisms of human FcγR genes include all those known inthe art. For the human FcγRIIA gene, polymorphisms include, e.g., thehigh responder and low responder phenotype reported by the ability of Tcells to proliferate in response to IgG. The high responder polymorphismis characterized by an arginine residue at position 131 (131Arg) whilethe low responder is characterized by a histidine residue at position131 (131His). In a specific embodiment, the human FcγRIIA sequencecomprises the 131His polymorphism. A representative protein sequence ofthe human FcγRIIA α-chain is shown in SEQ ID NO:32.

Single-nucleotide substitutions of the human FcγRIIB gene result inmis-sense substitutions in the ligand-binding domain (α-chain) andputatively affect the binding ability of an Fc portion of an IgG to bindto the α-chain of FcγRIIB on the cell surface. For example, substitutionof a threonine residue for an isoleucine at position 232 (Ile232Thr)within the transmembrane domain of the FcγRIIB gene in mice has beenshown to impair the signaling ability of the receptor. In a specificembodiment, the human FcγRIIB gene comprises the isoleucine variant(232Ile). A representative protein sequence of the human FcγRIIB α-chainis shown in SEQ ID NO:33.

Allelic variants of the human FcγRIIIA gene are proposed to be involvedin susceptibility to SLE and RA. This allelic variant includes aphenylalanine substitution for valine at position 158 (Val158Phe). Thevaline allelic variant (158Val) is characterized to have a higheraffinity for IgG1 and IgG3 than the phenylalanine allelic variant(158Phe). The 158Phe allelic variant has been proposed to lead to areduced clearance of immune complexes. In a specific embodiment, thehuman FcγRIIIA gene comprises the 158Val allelic variant. Arepresentative protein sequence of the human FcγRIIIA α-chain is shownin SEQ ID NO:35.

Allelic variants of the human FcγRIIIB gene include the neutrophilantigen 1 (NA1) and neutrophil antigen 2 (NA2) alleles. These allelicvariants have been proposed to be involved in blood-transfusionreactions, alloimmune neutropaenia, SLE and Wegener's granulomatosis.The NA2 allelic variant is characterized by a diminished ability tomediate phagocytosis. In a specific embodiment, the human FcγRIIIB genecomprises the NA2 allelic variant. A representative protein sequence ofthe human FcγRIIIB α-chain is shown in SEQ ID NO:36.

In one aspect, the genetically modified non-human animals are useful foroptimizing FcγR-mediated functions triggered by the Fc portion oftherapeutic antibodies. The Fc regions of antibodies can be modified byany method known in the art. For example, amino acid residues within theFc portion (e.g., CH2 and CH3 domains) can be modified to selectivelyenhance the binding affinity to human FcγRIIIA. Thus, the resultingantibody should have enhanced FcγRIIIA-dependent ADCC. In a specificembodiment, an animal expressing human FcγRIIIA of the present inventionis used to evaluate the enhanced ADCC ability of a modified humanantibody by administering a modified human antibody to the animal,detecting (e.g., in vitro) antibody binding to FcγRIIIA-expressing cellsand comparing the ADCC activity observed to the ADCC activity observedfrom that determined in a wild type animal.

EXAMPLES Example 1 Generation of Low Affinity FcγR Gene Deficient Mice

A targeting construct for introducing a deletion of the endogenous lowaffinity mouse FcγR locus (described below) was constructed (FIG. 1).

The targeting construct was made using VELOCIGENE® technology (see,e.g., U.S. Pat. No. 6,586,251 and Valenzuela et al. (2003)High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotech. 21(6):652-659) tomodify the Bacterial Artificial Chromosome (BAC) RP23-395f6(Invitrogen). RP23-395f6 BAC DNA was modified to delete the endogenouslow affinity FcγRIIB, FcγRIV and FcγRIII genes comprising the α-chain ofeach of the FcγRs.

Briefly, upstream and downstream homology arms were made employingprimers mFcR 5-up-1 (5′-ACCAGGATAT GACCTGTAGA G; SEQ ID NO:1) and mFcR3-up-1a (GTCCATGGGT AAGTAGAAAC A; SEQ ID NO:2), and mFcR 5-DN(ATGCGAGCTC ATGCATCTATG TCGGGTGCGG AGAAAGAGGT AATGCATTCT TGCCCAATACTTAC; SEQ ID NO:3) and mFcR 3-DN (ACTCATGGAG CCTCAACAGG A; SEQ ID NO:4),respectively. These homology arms were used to make a cassette thatdeleted the α-chains of the endogenous low affinity FcγRIIB, FcγRIV andFcγRIII genes. The targeting construct included a loxed neomycinresistance gene comprising homology arms comprising sequence homologousto a 5′ and a 3′ region with respect to the endogenous locus. Genesand/or sequences upstream of the endogenous FcγRIIB gene and downstreamof the endogenous FcγRIII gene (see FIG. 1) were unmodified by thetargeting construct.

The targeted deletion was confirmed by polymerase chain reaction (PCR)using primers outside the deleted region and within the targetingconstruct. The upstream region of the deleted locus was confirmed by PCRusing primers to mFcR-up-detect (ATCCTGAGTA TACTATGACA AGA; SEQ ID NO:5)and PGK-up-detect (ACTAGTGAGA CGTGCTACTT C; SEQ ID NO:6), whereas thedownstream region of the deleted locus was confirmed using primerspA-DN-detect (CTCCCACTCA TGATCTATAG A; SEQ ID NO:7) and mFcR-DN-detect(TGGAGCCTCA ACAGGACTCC A; SEQ ID NO:8). The nucleotide sequence acrossthe upstream deletion point included the following, which indicatesendogenous mouse sequence downstream of the FcγRIIB gene (containedwithin the parentheses below) linked contiguously to cassette sequencepresent at the deletion point: (GTCCATGGGT AAGTAGAAAC A)TTCGCTACCTTAGGACCGT TA (SEQ ID NO:9). The nucleotide sequence across thedownstream deletion point included the following, which indicatescassette sequence contiguous with endogenous mouse sequence upstream ofthe FcγRIII gene (contained within the parentheses below): CGGGTGCGGAGAAAGAGGTA AT(GCATTCTT GCCCAATACT TA) (SEQ ID NO:10).

Mice deficient in FcγRIIB, FcγRIII and FcγRIV were generated throughelectroporation of a targeted BAC DNA (described above) into mouse EScells. Positive ES cells clones are confirmed by Taqman™ screening andkaryotyping. Positive ES cell clones were then used to implant femalemice to give rise to a litter of pups deficient in low affinity FcγRgenes.

Example 2 Characterization of Low Affinity FcγR Gene Deficient Mice

Spleens were harvested from FcγR deficient and wild type mice andperfused with 10 mL Collagenase-D in sterile disposable bags. Each bagcontaining a single spleen was then placed into a Stomacher® (Seward)and homogenized at a medium setting for 30 seconds. Homogenized spleenswere transferred to 10 cm petri dishes and incubated for 25 minutes at37° C. Cells were separated with a pipette using a 1:50 dilution of 0.5M EDTA, followed by another incubation for five minutes at 37° C. Cellswere then pelleted with a centrifuge (1000 rpm for 10 minutes) and redblood cells were lysed in 4 mL ACK buffer (Invitrogen) for threeminutes. Splenocytes were diluted with RPMI-1640 (Sigma) and centrifugedagain. Pelleted cells were resuspended in 10 mL RPMI-1640 and filteredwith a 0.2 μm cell strainer.

Flow Cytometry

Lymphocyte cell populations were identified by FACs on the BD LSR IISystem (BD Bioscience) with the following flourochrome conjugated cellsurface markers: anti-CD19 (B cells), anti-CD3 (T cells), anti-NKp46 (NKcells) and anti-F4/80 (macrophages). Lymphocytes were gated for specificcell lineages and analyzed for expression of endogenous FcγRIII andFcγRIIB with a rat anti-mouse FcγRIII/II antibody (clone 2.4G2, BDBiosciences). Clone 2.4G2 recognizes a common polymorphic epitope on theextracellular domains of murine FcγRIII and FcγRII. The results showthat there was no detectable murine low affinity FcγRIII or FcγRII onB-cells, NK cells and macrophages in mFcγR KO mice (FIG. 2).

ADCC Assay

Splenocytes isolated from FcγR gene deficient and wild type mice wereanalyzed for their ability to perform ADCC in a cell-killing assay. Cellpopulations were isolated and separated using MACS® Technology (MiltenyiBiotec). Briefly, T-cells were depleted from splenocytes usingmagnetically labeled anti-mouse CD3 beads. The T-cell depletedsplenocytes were then enriched for NK cells using magnetically labeledanti-mouse CD49B beads. Separately, Raji cells (expressing human CD20)were coated with varying concentrations (ranging from 0.1 to 10 μg/mL)of mouse anti-human CD20 antibody (Clone B1; Beckman Coulter) for 30minutes at 4° C. The antibody-coated Raji cells were incubated with theenriched NK cells at ratios (NK:Raji) of 100:1 and 50:1 for four hoursat 37° C. Cell death was measured using the CytoTox-Glo™ CytotoxicityAssay (Promega). Luminescence signal is derived from lysed cells andproportional to the number of dead cells. Luminescence from controls (noanti-CD20 antibody) was determined for background dead cell count foreach ratio and subtracted from measurements for wild type and KO mice.Average cell death was calculated and percent decrease in cell killing(% ADCC) was determined by comparison to wild type. Results are shown inTable 1.

TABLE 1 % ADCC 10 μg/mL 1 μg/mL 0.1 μg/mL mFcγR KO B1 Antibody B1Antibody B1 Antibody NK cell:Raji cell 100:1 42 53 35  50:1 15 0 0

Example 3 In Vivo Depletion of B cells in Low Affinity FcγR GeneDeficient Mice

The effect of human or murine Fc isotypes on B cell depletion throughthe ADCC pathway was determined for various B cell compartments in lowaffinity FcγR gene deficient mice engineered to express human CD20 usinga human anti-human CD20 antibody. Mice expressing human CD20 wereseparately engineered using techniques known in the art. Mice thatexpress human CD20 on B cells and deficient in low affinity FcγR genes(described in Example 1) were made by standard breeding techniques ofthe two engineered strains.

Separate groups of mice that expressed human CD20 and had a fullcomplement of endogenous low affinity FcγR genes were each administeredone of the following: (1) 10 mg/kg control antibody (N=4; human antibodynot specific for human CD20 having a mouse IgG2a); (2) 2 mg/kg Ab 168(N=3; human anti-hCD20 antibody with a mouse IgG2a; heavy and lightchain variable region sequences found in SEQ ID NOs: 339 and 347,respectively, of US Patent Publication No. 2009/0035322); (3) 10 mg/kgAb 168; (4) 2 mg/kg Ab 735 (N=3; Ab 168 with human IgG1); (5) 10 mg/kgAb 735. In a similar set of experiments, groups of mice that expressedhuman CD20 and had a deletion of the endogenous low affinity FcγR geneswere administered the control and human anti-hCD20 antibodies (describedabove).

Mice in each group were administered the antibodies by intra-peritonealinjections. Seven days post-injection, animals were euthanized and theremaining B cell contents of bone marrow (B220⁺/IgM⁺), peripheral blood(B220⁺/CD19⁺), lymph node (B220⁺/CD19⁺) and spleen (B220⁺/CD19⁺) wereidentified by multi-color FACS performed on a LSR-II flow cytometer andanalyzed using Flow-Jo software (as described above). The results of theB cell depletion experiments are shown in FIGS. 3A-3D.

As shown in FIGS. 3A-3D, Ab 735 depleted B cells with a lower efficiencythan Ab 168 in mice containing a complete complement of low affinityFcγR genes. Further, for both antibodies (mouse and human Fc), B celldepletion was significantly reduced in mice lacking a completecomplement of low affinity FcγR genes. This Example shows that theability to deplete B cells through the ADCC pathway requires lowaffinity FcγRs and demonstrate that measuring ADCC efficiency forantibodies containing human constant regions in mice is more suitable bythe use of genetically engineered mice containing a full complement ofhuman low affinity FcγR genes.

Example 4 Generation of FcγRIIIA/FcγRIIA Humanized Mice

A targeting construct for introducing two low affinity human FcγR genesinto a deleted endogenous low affinity mouse FcγR locus (describedbelow) was constructed (FIG. 4).

A targeting construct comprising human FcγRIIA and FcγRIIIA genes wasmade using similar methods (see Example 1) through modification of BACRP23-395f6 and CTD-2514j12 (Invitrogen). BAC DNA of both BACs wasmodified to introduce a deletion of the α-chains of the low affinityhuman FcγRIIA and FcγRIIIA genes into the deleted endogenous lowaffinity FcγR locus.

In a similar fashion, upstream and downstream homology arms were madeemploying primers h14 (GCCAGCCACA AAGGAGATAA TC; SEQ ID NO:11) and h15(GCAACATTTA GGACAACTCG GG; SEQ ID NO:12), and h4 (GATTTCCTAA CCACCTACCCC; SEQ ID NO:13) and h5 (TCTTTTCCAA TGGCAGTTG; SEQ ID NO:14),respectively. These homology arms were used to make a cassette thatintroduced the α-chains of low affinity human FcγRIIA and FcγRIIIA genesinto the endogenous mouse low affinity FcγR locus. The targetingconstruct included a 5′ homology arm including sequence 5′ to thedeleted endogenous low affinity FcγR locus, a FRT'ed hygromycinresistance gene, followed by a human genomic fragment from BACCTD-2514j12 comprising low affinity human FcγRIIA and FcγRIIIA α-chaingenes, and a 3′ homology arm comprising mouse sequence 3′ to the deletedendogenous low affinity FcγR locus (middle of FIG. 4). For a mouse thatexpresses FcγRIIA on mouse platelets, a targeting construct was made ina similar manner (using the same BACs) except that the constructcomprises an extended promoter sequence operably linked to the humanFcγRIIA gene in the human genome, e.g., up to about 18 kb or more, usinga hygromycin cassette that is flanked on both sides by lox2372 sites,wherein the junction of the promoter region and the first lox 2372 siteis ATCGGGGATA GAGATGTTTG (CC)GCGATCGC GGTACCGGGC (SEQ ID NO:37human/lox2372 junction in parentheses) and wherein the junction of thesecond lox2372 site and mouse sequence is TTATACGAAG TTATACCGG(TG)CATTCTTGC CCAATACTTA (SEQ ID NO:38 lox2372/mouse junction inparentheses). Suitable primers were used to genotype the humanizationcomprising the promoter region.

Targeted insertion of the human FcγRIIA and FcγRIIIA α-chain genes wasconfirmed by PCR (as described above). The upstream region of thepartially humanized locus was confirmed by PCR using primers h16(CCCAGGTAAG TCGTGATGAA ACAG; SEQ ID NO:15) and pA-DN-detect (CTCCCACTCATGATCTATAG A; SEQ ID NO:16), whereas the downstream region of thepartially humanized locus was confirmed using primers mFcR DN-detect-9(TGGAGCCTCA ACAGGACTCC A; SEQ ID NO:17) and h6 (CACACATCTC CTGGTGACTT G;SEQ ID NO:18). The nucleotide sequence across the downstream junctionincluded the following, which indicates a novel insertion point ofendogenous human sequence upstream of the hFcγRIIA gene (containedwithin the parentheses below) contiguous with endogenous mouse sequence3′ of the deleted low affinity FcγR locus: (CAACTGCCAT TGGAAAAGA)CTCGAGTGCCA TTTCATTACC TC (SEQ ID NO:19). The upstream junction includestwo novel sequences. One point of the upstream junction includes thefollowing, which indicates nucleotide sequence of the hygromycincassette contiguous with human genomic sequence (contained within theparentheses below) that comprises the upstream region of the insertedhFcγRIIIA gene: TAAACCCGCG GTGGAGCTC(G CCAGCCACAA AGGAGATAAT CA) (SEQ IDNO:20). The second point of the upstream junction includes thefollowing, which indicates a nucleotide sequence of an endogenous mousesequence (contained within the parentheses below) from the upstreamregion of the deleted low affinity FcγR locus contiguous with anucleotide sequence within the hygromycin cassette: (CCATGGGTAAGTAGAAAC)TC TAGACCCCCG GGCTCGATAA CT (SEQ ID NO:21).

Mice containing two low affinity human FcγR genes (hFcγRIIA, lackingextended promoter region, and hFcγRIIIA) in place of the endogenous lowaffinity mouse FcγR locus were generated through electroporation of thetargeted BAC DNA (described above) into mouse ES cells. Positive EScells clones were confirmed by Taqman™ screening and karyotyping.Positive ES cell clones were then used to implant female mice using theVELOCIMOUSE® method (described below) to generate a litter of pupscontaining a replacement of the endogenous low affinity FcγR genes withthe two human low affinity FcγR genes.

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99. VELOCIMICE® (F0 mice fully derived from the donorES cell) bearing hFcγRIIA and hFcγRIIIA were identified by genotypingusing a modification of allele assay (Valenzuela et al., supra) thatdetected the presence of the hFcγR genes.

Mice bearing the hFcγR genes can be bred to a Cre deleter mouse strain(see, e.g., International Patent Application Publication No. WO2009/114400) in order to remove any loxed neo cassette introduced by thetargeting construct that is not removed, e.g., at the ES cell stage orin the embryo. Optionally, the neomycin cassette is retained in themice.

Pups are genotyped and a pup heterozygous for the hFcγR genes isselected for characterizing FcγRIIA and FcγRIIIA humanizations.

Example 5 Characterization of FcγRIIIA/FcγRIIA Humanized Mice

Spleens were harvested from humanized FcγRIIIA/FcγRIIA (heterozygotes,lacking the extended FcγRIIA promoter region) and wild type mice andprepared for FACs (as described above).

Flow Cytometry

Lymphocytes were gated for specific cell lineages and analyzed forexpression of hFcγRII and hFcγRIII using a mouse anti-human FcγRIIantibody (Clone FLI8.26; BD Biosciences) and a mouse anti-human FcγRIIIantibody (Clone 3G8; BD Biosciences), respectively. Relative expression(++, +) or no expression (−) observed for each lymphocyte subpopulationis shown in Table 2.

TABLE 2 Lymphocyte Lineage hFcγRIII hFcγRII B cells − − NK cells ++ −Macrophages + + Neutrophils − +

In a similar experiment, spleens were harvested from humanizedFcγRIIIA/FcγRIIA (homozygotes, lacking the extended FcγRIIA promoterregion) and wild type mice and prepared for FACs (as described above).Results are shown in FIGS. 5A and 5B. Percent of separate lymphocytecell populations expressing human FcγRIIIA, FcγRIIA or both inFcγRIIIA/FcγRIIA homozygote mice is shown in Table 3.

TABLE 3 Lymphocyte Lineage hFcγRIII hFcγRII hFcγRII/hFcγRIII NK cells 97— — Macrophages 26 14 39 Neutrophils — 94 —

As shown in this Example, genetically modified mice (both heterozygoteand homozygote genotypes) generated in accordance with Example 3expressed human FcγRIIIA on NK cells and macrophages; and human FcγRIIAon neutrophils and macrophages, but not platelets. Human FcγRIIIA washighly expressed on NK cells. The expression pattern of human FcγR genesshown in this Example is consistent with the expression patterns ofthese genes in human accessory cells.

Example 6 Generation of Low Affinity FcγR Humanized Mice

A targeting construct for introducing three additional low affinityhuman FcγR genes into a partially humanized endogenous low affinity FcγRlocus (described below) was constructed (FIG. 6).

A targeting construct comprising human FcγRIIB, FcγRIIIB and FcγRIICgenes was made using similar methods (see Example 1) throughmodification of BAC RP-23 395f6 and RP-11 697e5 (Invitrogen). BAC DNA ofboth BACs was modified to introduce the α-chains of the low affinityhuman FcγRIIB, FcγRIIIB and FcγRIIC genes into the partially humanizedendogenous low affinity FcγR locus containing two human low affinityFcγR genes.

In a similar fashion, upstream and downstream homology arms were madeemploying primers mFcR up-1 (ACCAGGATAT GACCTGTAGA G; SEQ ID NO:22) andmFcR2b Nhel-2 (GTTTCTACTT ACCCATGGAC; SEQ ID NO:23), and h10 (AAATACACACTGCCACAGAC AG; SEQ ID NO:24) and h11 (CCTCTTTTGT GAGTTTCCTG TG; SEQ IDNO:25), respectively. These homology arms were used to make a cassettethat introduced DNA sequences encoding the α-chains of low affinityhuman FcγRIIB, FcγRIIIB and FcγRIIC. The targeting construct included a5′ homology arm including mouse sequence 5′ to the deleted endogenouslow affinity FcγR locus, a loxed neomycin resistance gene, followed by ahuman genomic fragment from BAC RP-11 697e5 comprising low affinityhuman FcγRIIB, FcγRIIIB and FcγRIIC α-chain genes, and a 3′ homology armcomprising human sequence 5′ to the low affinity human FcγRIIIA α-chaingene (middle of FIG. 6).

Targeted insertion of three additional low affinity human FcγR genes wasconfirmed by PCR (as described above). The upstream region of the fullyhumanized locus was confirmed by PCR using primers mFcR up-detect-3(GAGTATACTA TGACAAGAGC ATC; SEQ ID NO:26) and PGK up-detect (ACTAGTGAGACGTGCTACTT C; SEQ ID NO:27), whereas the downstream region of the fullyhumanized locus was confirmed using primers neo detect (CTCCCACTCATGATCTATAG A; SEQ ID NO:28) and h12 (CTTTTTATGG TCCCACAATC AG; SEQ IDNO:29). The nucleotide sequence across the downstream junction includedthe same human genomic sequence upstream of the hFcγRIIA α-chain gene(see Example 3; SEQ ID NO:19). The nucleotide sequence across theupstream junction included the following, which indicates two noveljunctions of mouse and cassette sequences and cassette and human genomicsequences at the insertion point. The junction of genomic mouse sequence(contained within the parentheses below) and the upstream region of theneo cassette sequence is: (GTCCATGGGT AAGTAGAAAC A)TTCGCTACC TTAGGACCGTTA (SEQ ID NO:30). The second novel junction includes the joining of the3′ end of neo cassette (contained within the parentheses below) and ahuman genomic sequence downstream of the hFcγRIIB α-chain gene:(GCTTATCGAT ACCGTCGAC)A AATACACACT GCCACAGACA GG; SEQ ID NO:31). Thesejunctions are show in FIG. 6 (middle) within the targeting construct.The resulting modified genome of the fully humanized low affinity FcγRlocus is shown in FIG. 6 (bottom).

Mice containing five low affinity human FcγR genes in place of theendogenous low affinity mouse FcγR locus were generated throughelectroporation of the targeted BAC DNA (described above) into mouse EScells. Positive ES cells clones were confirmed by Taqman™ screening andkaryotyping. Positive ES cell clones were then used to implant femalemice (as described above) to give rise to a litter of pups containing areplacement of the endogenous low affinity FcγR genes for five human lowaffinity FcγR genes.

Example 7 Characterization of Low Affinity FcγR Humanized Mice

Spleens were harvested from fully humanized FcγR (heterozygotes) andwild type mice and prepared for FACs (as described above).

Flow Cytometry

Lymphocytes were gated for specific cell lineages and analyzed forexpression of human FcγRIIA and FcγRIIIA using a mouse anti-human FcγRIIantibody (Clone FLI8.26; BD Biosciences) and a mouse anti-human FcγRIIIantibody (Clone 3G8; BD Biosciences), respectively. Relative expression(++, +) or no expression (−) observed for each lymphocyte subpopulationis shown in Table 4.

TABLE 4 Lymphocyte Lineage hFcγRIII hFcγRII B cells − + NK cells + +Macrophages + + Neutrophils + +

In a similar experiment, spleens were harvested from fully humanizedFcγR (homozygotes) and wild type mice and prepared for FACs (asdescribed above). Results are shown in FIG. 7. Percent of separatelymphocyte cell populations expressing human FcγRIIIA, human FcγRIIIB,human FcγRIIA, human FcγRIIB, human FcγRIIC or a combination thereof infully humanized FcγR homozygote mice is shown in Table 5.

TABLE 5 Lymphocyte Lineage hFcγRIII hFcγRII hFcγRII/hFcγRIII B cells 100NK cells 30 — — Macrophages <1 55 26 Neutrophils — 100

As shown in this Example, genetically modified mice (both heterozygoteand homozygote genotypes) generated in accordance with Example 5expressed human FcγRIIIA on NK cells and macrophages, human FcγRIIIB onneutrophils, human FcγRIIA on neutrophils and macrophages, human FcγRIIBon B cells, and human FcγRIIC on NK cells. The expression pattern ofhuman FcγR genes shown in this Example is consistent with the expressionpatterns of these genes in human accessory cells.

Example 8 ADCC in Humanized FcγR Mice

Splenocytes isolated from FcγR gene deficient (i.e. knockout),FcγRIIIA/FcγRIIA (homozygotes),FcγRIIIA/FcγRIIIB/FcγRIIA/FcγRIIB/FcγRIIC (homozygotes) and wild typemice were analyzed for their ability to perform ADCC in a cell-killingassay (as described above in Example 2).

Briefly, cell populations were isolated and separated using MACS®Technology (Miltenyi Biotec). Briefly, T and B cell depleted splenocyteswere cultured for two weeks in the presence of mouse IL-2 (500 U/mL).The resulting expanded NK cells were used as effector cells in the ADCCassays at a ratio of 50:1 (NK:Raji). Raji cells were coated with 10ug/mL of Ab 168 or Ab 735 (as described above in Example 3). Results areshown in Table 6.

TABLE 6 % ADCC 10 μg/mL 10 μg/mL NK Cell Genotype Ab 168 Ab 735 WildType 89 72 Mouse FcγR KO 13 14 Human FcγRIIIA-IIA HO 78 85 HumanFcγRIIIA-IIIB-IIA-IIB-IIC HO 81 59

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

We claim:
 1. A genetically modified mouse that does not express anendogenous FcγRIIB α-chain, an endogenous FcγRIV α-chain, and anendogenous FcγRIII α-chain.
 2. The mouse of claim 1, wherein the mousecomprises a reduced ability to make an immune response to an antigen ascompared to a wild-type mouse with respect to the same antigen.
 3. Themouse of claim 1, wherein the mouse comprises at least a 50% reductionin antibody-dependent cell-mediated cytotoxicity (ADCC).
 4. The mouse ofclaim 1, wherein the mouse expresses a functional FcR γ-chain.
 5. Themouse of claim 1, wherein the mouse further expresses human CD20.
 6. Agenetically modified mouse comprising a deletion of an endogenousFcγRIIB α-chain gene, an endogenous FcγRIV α-chain gene, and a deletionof an endogenous FcγRIII α-chain gene.
 7. The mouse of claim 6, whereinthe mouse comprises a reduced ability to make an immune response to anantigen as compared to a wild-type mouse with respect to the sameantigen.
 8. The mouse of claim 6, wherein the mouse comprises at least a50% reduction in antibody-dependent cell-mediated cytotoxicity (ADCC).9. The mouse of claim 6, wherein the mouse comprises a functional FcRγ-chain gene.
 10. The mouse of claim 6, wherein the mouse furthercomprises a humanized CD20 gene.
 11. A genetically modified non-humancell comprising a deletion of an endogenous FcγRIIB α-chain gene, anendogenous FcγRIV α-chain gene, and a deletion of an endogenous FcγRIIIα-chain gene.
 12. The cell of claim 11, wherein the cell is a mousecell.
 13. The cell of claim 11, wherein the cell is a rat cell.
 14. Thecell of claim 11, wherein the cell comprises a functional FcR γ-chain.15. The cell of claim 12, wherein the cell is an embryonic stem (ES)cell.
 16. The cell of claim 12, wherein the cell is a natural killer(NK) cell.
 17. The cell of claim 13, wherein the cell is an ES cell. 18.The cell of claim 11, wherein the cell comprises a human CD20 gene. 19.A method of making a genetically modified non-human animal that does notexpress an endogenous FcγRIIB α-chain, an endogenous FcγRIV α-chain, andan endogenous FcγRIII α-chain, comprising using the cell of claim 11.20. A method of making a genetically modified mouse that does notexpress an endogenous FcγRIIB α-chain, an endogenous FcγRIV α-chain, andan endogenous FcγRIII α-chain, comprising using the cell of claim 15.